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bioRxiv · 10.1101/2022.11.22.516823

Precision super-resolution cryo-correlative light and electron microscopy for rapid in situ structural analyses of optogenetically-positioned organelles

Redpath, G. M. I.·Rae, J. A.·Yao, Y.·Ruan, J.·Cagigas, M. L.·Whan, R.·Hardeman, E. C.·Gunning, P. W.·Ananthanarayanan, V.·Parton, R. G.·Ariotti, N.
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Abstract

Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a novel cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes in the cell by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. We have developed a protocol where cells can be frozen, imaged by cryo-fluorescence microscopy and ready for batch cryo-ET within a day.

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Redpath, G. M. I., Rae, J. A., Yao, Y., Ruan, J., Cagigas, M. L., Whan, R., Hardeman, E. C., Gunning, P. W., Ananthanarayanan, V., Parton, R. G., Ariotti, N.. 2022-11-23. Precision super-resolution cryo-correlative light and electron microscopy for rapid in situ structural analyses of optogenetically-positioned organelles. https://doi.org/10.1101/2022.11.22.516823

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